The role of phosphorous in animal nutrition is well recognized, it is a critical component of the skeleton, nucleic acids, cell membranes and some vitamins. Though phosphorous is essential for the health of animals, not all phosphorous in feed is bioavailable.
Phytates are the major form of phosphorous in seeds, for example phytate represents about 60-80% of total phosphorous in corn and soybean. When seed-based diets are fed to non-ruminants, the consumed phytic acid forms salts with several important mineral nutrients, such as potassium, calcium, and iron, and also binds proteins in the intestinal tract. These phytate complexes cannot be metabolized by monogastric animals and are excreted, effectively acting as anti-nutritional factors by reducing the bioavailability of dietary phosphorous and minerals. Phytate-bound phosphorous in animal excreta also has a negative environmental impact, contributing to surface and ground water pollution.
There have been two major approaches to reducing the negative nutritional and environmental impacts of phytate in seed. The first involves post-harvest interventions, which increase the cost and processing time of feed. Post-harvest processing technologies remove phytic acid by fermentation or by the addition of compounds, such as phytases.
The second is a genetic approach, which has been strongly correlated with undesirable agronomic characteristics. One genetic approach involves developing crop germplasm with heritable reductions in seed phytic acid. Heritable quantitative variation in seed phytic acid has been observed among lines in several crop species, but is also highly and positively correlated with less desirable characteristics. While some variability for phytic acid was observed, there was no change in non-phytate phosphorous, only 2% of the observed variation in phytic acid was heritable whereas 98% of the variation was attributed to environmental factors.
Another traditional genetic approach involves selecting low phytate lines from a mutagenized population to produce germplasm separated from the undesirable correlated traits seen in traditional breeding. Most mutant lines are a loss of function, presumably blocked in the phytic acid biosynthetic pathway; therefore low phytic acid accumulation will likely be a recessive trait. In certain cases, this approach has revealed that homozygosity for substantially reduced phytate proved lethal.
A more modern genetic approach is transgenic technology, which has been used to increase phytase levels in plants. These transgenic plant tissues or seed have been used as dietary supplements, but this approach has not been used to reduce phytic acid accumulation in seed.
The biosynthetic route leading to phytate is complex and not completely understood. Without wishing to be bound by any particular theory of the formation of phytate, it is believed that the synthesis may be mediated by a series of one or more ADP-phosphotransferases, ATP-dependent kinases and isomerases. A number of intermediates have been isolated including, for example, monophosphates such as D-myo-inositol 3-monophosphate, diphosphates (IP2s) such as D-myo-inositol 3,4-bisphosphate, triphosphates (IP3s) such as D-myo-inositol 3,4,6 trisphosphate, tetraphosphates (IP4s) such as D-myo-inositol 3,4,5,6-tetrakisphosphates, and pentaphosphates (IP5s) such as D-myo-inositol 1,3,4,5,6 pentakisphosphate. The phosphorylation of the IP5 to IP6 is found to be reversible. Several futile cycles of dephosphorylation and rephosphorylation of the P5 and P6 forms have been reported as well as a cycle involving glucose-6-phosphate->D-myo-inositol 3-monophosphate->myo-inositol; the last step being completely reversible, indicating that control of metabolic flux through this pathway may be important.
Based on the foregoing, there exists the need to improve the nutritional content of plants, particularly corn and soybean by increasing non-phytate phosphorous and reducing seed phytate. This invention differs from the foregoing approaches in that it provides tools and reagents that allow the skilled artisan, by the application of, inter alia, transgenic methodologies to influence the metabolic flux in respect to the phytic acid pathway.